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Biophysik der Molekle

4. Vorlesung Rdler WS 2010

28. Oct. 2010

Protein folding- Afinsen hypothesis- hydrophobic interaction

Gaub/SS 2005 BPM 1.3 2

Protein Unfolding: Sushi Restaurant

When foods with proteins are exposed to heat and certain chemicals (such as vinegar), they turn white.

1. Distinguish salmon roe from imitation salmon

roe by dropping into hot tea.

2. Mackerel is pickled in vinegar for

preservation.

Gaub/SS 2005 BPM 1.3 3

Nobel Prize for Chemistry in 1972

C. Afinsen 1916-1995

The Thermodynamic Hypothesis (Afinsen 1973)

the native state is thermodynamically stable

=> the sequence alone

determines 3D structure!

ribonuclease A

loop(usually exposed on surface)

alpha-helix beta-sheet

Afinsensmodel protein:ribonuclease A

Ribonuclease kann durch Oxidation (Spaltung der S-S Bindung)denaturiert werden

o) Nofr t iche

Ribonuclea se

Abb. 3.12: Die zwei Zuslnde der Ribonuklease:

links: KomDakt! Funktionsfom

rechts: Oenatudert! Form

b )0eno tu r ie r te

Ribonu cleose95

HH

65

Das Enzym hat 8 s-s Bindungen. Im Prinzip knnten 56 verschiedene Zustnde (Isomere) gebildet werden. Es gibt aber offenbar nur einen Zustand niedrigster Energie.

7

Folding of RNAse A in the test tube

denaturation renaturation

Incubate proteinin guanidine

hydrochloride(GuHCl)or urea

100-folddilution of proteininto physiological

buffer

Anfinsen, CB (1973) Principles that govern the folding of protein chains.

Science 181, 223-230.

- the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation

Thus: protein folding is a spontaneous process that does not require the assistance of extraneous factors

(aggregation)

Folding of proteins in vivo is promoted by chaperones

this bears only on the rate of folding

However:

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What drives protein folding?

Minimization of G=E-TS+PV

Minimize the solvation energy.Decrease the conformational entropy.

10

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GFP Fluoreszenz Siehe Biophysik F-Praktikum

Other techniques to probe unfolding

High-resolution techniques (local):

FTIR

Fluorescence

NMR

UV absorption

Low-resolution techniques:

SAXS

DLS

Which forces are dominant in protein folding ?Local vs. non-local interactions

Nonlocal interactions drive collapse transition, whereas local interactions drive helix transitions.

16

Early model in which protein folding was proposed to be

driven by ion-paired hydrogen bonding among side chains

(Mirsky& Pauling, 1936; Eyring & Stearn, 1939)

disproven by Jacobsen and Linderstrom-Lang

Electrostatic Contributions

!i=(zie/4"#o)(1/r2) coulomb potential

Sensitive to pH and ion concentrations

pH determines total charge (pI)

Ionic strength determines effective range of interactions

Ion pairs contribute 1-3 kcal/mol (on surface)

Ion pairs generally destabilizing if buried (cost up to 19 kcal/mol/ion to completely bury

Ion pairs contribute ~5-15 kcal/mol per 150 aas

The Kauzmann Hypothesis

hydrophobic interactions determine the thermal stability of the native state

* non-polar solvents denature proteins* unusual temperature dependence: (stability decreases at high AND low temperatures)* protein stability follows same salt dependence as lyotropic (Hofmeister) series

Key arguments

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Determination of protein stability.

This can be measured with a variety of tools including, microcalorimetry, spectroscopy, and enzyme function.

The transition can be accomplished with heat or denaturants.

The area under the curve gives $H which agrees with measurements based on the van't Hoff equation

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Denaturants Temperature

pH Change the ionization state of critical residues

Detergents Bind strongly to the unfolded protein

High concentrations of water soluble organic substances Aliphatic alcohols. These disrupt the water structure

Ionic or polar denaturants including urea and guanidinium

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Denaturants: The Hofmeister Series

The ability of an ion to stabilize a protein follows the Hofmeister series

Anions! SO42->H2PO4->CH3COO->Cl->Br->I'->ClO4->SCN-

Cations

NH4+,Cs+,K+,Na+>Li+>Mg2+>Ca2+>Ba2+ ! ! ! >guanidinium>urea

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Thermal stability of RNase A as a

function of salt

This illustrates the effect on protein stability for many commonly used salts.

Potassium phosphate and ammonium sulfate stabilize proteins which accounts for their frequent use in protein purification.

From Voet and Voet second edition

The hydrophobic effect

water forms cluster with coordination number 4

proteins are surrounded bya shell of structured water

!

K ,W

= K ,W

0 + RT ln xK ,W

Solubility and partition function

!

K

0"

W

0 = HK

0"H

W

0( ) "T SK0 " SW0( )!

W

= K

!

ln xW

* =K

0"

W

0( )RT

chemical potential,!, and partition coefficient,x of oil molecule in water (w) and oil (K)

at equilibrium:

!

" = W

0#

K

0 = 2.44 + 0.88nC

enthalpic entropic

[kcal/Mol]

The entropic change (cost of inducing water order) dominates over the enthalpy change (gain in intermolecular interaction), which is also negative.

!

" = #4.2 + 0.825nC

for alcoholes

for alcanes

!"#$%&'()*%+,-"#./*0#).1&).2&"3#)4*,,#$,0&5#).+).6*,,#$.7#+.89:.;

StoffW

0

! K

0

in kcal/Mol

HW

0! H

K

0

in kcal/Mol

SW

0! S

K

0

in cal/Mol K

C2H

63.9 -2.5 -21

C3H

84.9 -1.7 -22

C4H

105.9 -0.8 -23

HW

0

! HK

0!"#$%&'$()*$+,-$.&%$/%&$.%-$0/%-+,1-2'3$%&'%"$(45"$.%"$67$84'$9$'):1$;$+-%&;%-.%'.%$7$?)$.&%"%-$7%-#$'%3)@8$&"#A$&"#$.&%$0/%-+,1-2'3$%&'$%B4#1%-=%-$C-4D%*>$?&%$E)'F.%-F7))5"FG'D&%12'3$D;&":1%'$.%=$67F(45%9,5$2'.$7)""%-$&"#$/%#-$

$!"#$'%3)@8A$.)"$/%.%2#%#A$%"$H'.%#$%&'%$G/')1=%$.%-$I)-@%55%'$J'#-4I&%$/%&$.%-

0/%-+,1-2'3$.%"$67$84'$9$'):1$;$"#)K>$LJ&"/%-3"#-29#2-$.%"$7)""%-"MSW0

! SK

0

! = W

0"

K

0= 2.44 + 0.88n

C%=I&-&":1N ':NO)15$.%-$6415%'"#4P)#4=%

Hydrophobic Effect At normal temps the hydrophobic effect is entropic

water molecules form ordered structures around nonpolar compounds

Hydrophobic residues collapse in to exclude water

Additional forces can then stabilize (vdw, h-bond,intrinsic properties)

Hydrophobic effect is dependent on temperature (unstable at high AND low temp).

Thermodynamic considerations Protein stability is composed of two components.

% % $G = $H-T$S

There is a complex temperature dependence for $H and T$S which means that the contribution of the enthalpic and entropic terms changes with temperature.

This temperature dependence arises from the anomalously high change in heat capacity on transferring hydrophobic compounds into water. This is the hall-mark of the hydrophobic effect and arises from the water-ordering.

Heat Capacity

The heat capacity influences both the temperature dependence of the enthalpy and entropy

It is proportional to the buried non-polar surface area as are all of the thermodynamic parameters.

The large heat capacity is indicative of a well ordered water structure around non-polar molecules in water as is evident from their partial specific volumes when dissolved in water

!

Cp ="H

"T=T"S

"T

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Temperature dependence of $G

Thermodynamics of transfer of a hydrocarbon from liquid to aqueous solution.

The temperature dependence is the result of different heat capacities of the two phases.

The large changes in $H and T$S compensate so that $G is fairly constant with temperature

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Temperature dependence of $H and T$S continued

$H becomes more favorable at lower temperatures, whereas the entropic term becomes less favorable. This is consistent with an increase in the order in the water surrounding the non-polar molecule.

The water-ordering increases the interaction between solvent and solute and thus "enhances" the solubility that would occur in its absence. Even so, the interactions between solute and water eliminate hydrogen bonds within the water that cannot be compensated for by the ordering of the water.

Significantly the van der Waals interactions are greater in the pure water and solute than in the dissolved solute.

It is the loss of hydrogen bonds and van der Waals interactions that is the cause of the hydrophobic interaction.

$H is ~0 at room temperature

Terms counterbalance

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Buried hydrophobic surface area The buried hydrophobic surface area for a protein

correlates with the protein stability.

Although it is difficult to predict the overall stability of a pr